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Preparation method for anti-mullerian hormone optical excitated chemiluminescent detection kit and kit and application thereof thereof

A technique for chemiluminescence detection and Müllerian hormone is applied in the field of preparation of anti-Müllerian hormone light-excited chemiluminescence detection kits, which can solve the problems of low sensitivity, narrow linear range, and easy environmental interference of detection methods.

Inactive Publication Date: 2018-12-11
上海铭源数康生物芯片有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In order to solve the above technical problems, one of the objects of the present invention is to provide a preparation method of an anti-Müllerian hormone light-excited chemiluminescence detection kit, which solves the problems of low sensitivity, narrow linear range and easily affected detection methods in the prior art. The problem of environmental interference

Method used

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  • Preparation method for anti-mullerian hormone optical excitated chemiluminescent detection kit and kit and application thereof thereof
  • Preparation method for anti-mullerian hormone optical excitated chemiluminescent detection kit and kit and application thereof thereof
  • Preparation method for anti-mullerian hormone optical excitated chemiluminescent detection kit and kit and application thereof thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0080] Example 1: Preparation steps of the luminescent reagent buffer of the kit (taking the preparation of 1L as an example, the formula is shown in Table 1)

[0081] ① Weigh 6g of HEPES into a 1L container, add an appropriate amount of purified water and stir,

[0082] ②Use a pipette to pipette 5mL of Triton X-100 into a beaker of 100mL of purified water, dissolve it completely and pour it into the above 1L container, add purified water to 900mL and stir thoroughly;

[0083] ③Adjust the pH meter to measure the pH of the solution, add HCl or NaOH to adjust the pH, so that it is controlled at 7.2-7.4;

[0084] ④Weigh 1g of casein and add it into the above 1L container, stir evenly to make it fully dissolved;

[0085] ⑤Weigh 1g of Dextran 500 and add it to the above 1L container, stir evenly to make it fully dissolved, ⑥Filtrate to 1L, filter with a 0.2μm filter, label it and store it in a sterile environment at 2-8°C.

[0086] Table 1 Luminescent Reagent Buffer Recipe

[00...

Embodiment 2

[0088] Example 2: The steps of coupling anti-AMH antibody to luminescent particles are as follows:

[0089] ① Preparation of luminescent particles: absorb a certain amount of luminescent particles and centrifuge in a high-speed refrigerated centrifuge, discard the supernatant, use a certain amount of 0.05M pH6.0MES buffer, ultrasonically break until the luminescent particles are resuspended, add 0.05M pH6.0MES The buffer adjusted the concentration of luminescent particles to 100mg / mL.

[0090] ② Antibody preparation: Add 100 μg of anti-AMH monoclonal antibody to the ultrafiltration tube, refrigerate and centrifuge at 10,000 g for 10 minutes, wash with 0.05M MES buffer at pH 6.0 for 5 times, and adjust the concentration of AMH antibody to 8 mg / mL .

[0091] ③Mix the 100mg / mL luminescent particle suspension prepared above with the 8mg / mL anti-AMH antibody at a mass ratio of 10:1, 20:1, 30:1, 40:1, 50:1, the best The mass ratio was 30:1, and quickly vortexed to obtain the react...

Embodiment 3

[0095] Example 3: Biotin-labeled anti-AMH antibody steps are as follows:

[0096] ① Antibody treatment: dialyze the anti-AMH antibody in 0.1M carbonic acid buffer, measure the antibody concentration and adjust it to 1mg / mL;

[0097] ② Prepare 16.17mg / mL biotin solution with DMSO;

[0098] ③Take the processed 1mg / mL anti-AMH antibody and the prepared biotin solution, mix according to the molecular ratio of biotin to antibody: 10:1, 20:1, 30:1, 40:1, 50:1 , the optimal molecular ratio is 30:1, quickly vortexed to mix. The reaction was shaken at room temperature for 4 hours.

[0099] ④Ultrafilter the reacted biotin-labeled antibody through an ultrafiltration tube to remove excess biotin.

[0100] ⑤ Aspirate the biotin-labeled antibody after ultrafiltration and transfer it to a clean centrifuge tube, take a sample to determine the antibody concentration, and adjust the biotin-labeled antibody concentration to 0.5 mg / mL with the luminescence reagent buffer of the kit.

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Abstract

The invention discloses a preparation method for an anti-mullerian hormone optical excitated chemiluminescent detection kit. The preparation method comprises the following steps: (1) preparing a luminescent reagent buffer solution; (2) preparing anti-AMH antibody coupled luminescent microparticles; (3) preparing a biotin labeled anti-AMH antibody; (4) preparing avidin labeled light-sensing microparticles; and (5) preparing an AMH calibrated product. The invention also discloses the anti-mullerian hormone optical excitated chemiluminescent detection kit prepared by the preparation method for the anti-mullerian hormone optical excitated chemiluminescent detection kit and an application method of the anti-mullerian hormone optical excitated chemiluminescent detection kit. By way of optical excitated chemiluminescence, that is chemiluminescence by means of singlet state oxygen transfer action between the light sensing microparticles and luminescent microparticles, the two microparticles are combined and luminescent, so that the detection specificity is improved. A luminescent material wrapped in the luminescent microparticles is unlikely to be interfered by the environment and has highspecificity and stability.

Description

technical field [0001] The present invention relates to the technical field of light-excited chemiluminescence detection of anti-Müllerian hormone (AMH) in serum (plasma), in particular to a preparation method of a light-excited chemiluminescence detection kit for anti-Mullerian hormone and its kit and Instructions. Background technique [0002] Anti-Mullerian hormone (AMH), also known as Mullerian inhibitor (Mullerian inhibiting substance, MIS), belongs to the transforming growth factor β (TGF-β) superfamily, and AMH is linked by disulfide bonds Glycoprotein dimer, with a relative molecular mass of 140 000, its coding gene is located on the short arm of chromosome 19, namely p13.2~p13.3, with 5 exons. [0003] AMH is secreted by immature Sertoli cells of the testis and granulosa cells of preantral follicles and small antral follicles of the ovary. AMH is a regulator of follicle growth and development. AMH participates in two important recruitments in the process of physio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/74G01N33/531G01N33/532G01N33/543
CPCG01N33/531G01N33/532G01N33/54313G01N33/74
Inventor 李亨芬施晓燕柳飞舟
Owner 上海铭源数康生物芯片有限公司
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